Characterization of nucleolar SUMO isopeptidases unveils a general p53-independent checkpoint of impaired ribosome biogenesis

Ribosome biogenesis is a multi-step process, in which a network of trans-acting factors ensures the coordinated assembly of pre-ribosomal particles in order to generate functional ribosomes. Ribosome biogenesis is tightly coordinated with cell proliferation and its perturbation activates a p53-dependent cell-cycle checkpoint. How p53-independent signalling networks connect impaired ribosome biogenesis to the cell-cycle machinery has remained largely enigmatic. We demonstrate that inactivation of the nucleolar SUMO isopeptidases SENP3 and SENP5 disturbs distinct steps of 40S and 60S ribosomal subunit assembly pathways, thereby triggering the canonical p53-dependent impaired ribosome biogenesis checkpoint. However, inactivation of SENP3 or SENP5 also induces a p53-independent checkpoint that converges on the specific downregulation of the key cell-cycle regulator CDK6. We further reveal that impaired ribosome biogenesis generally triggers the downregulation of CDK6, independent of the cellular p53 status. Altogether, these data define the role of SUMO signalling in ribosome biogenesis and unveil a p53-independent checkpoint of impaired ribosome biogenesis.

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A numerical value for number of cells or percentage (with statistics) is provided.with siRNA for 72 72 h.h.Cells were trypsinized and washed with PBS.Afterwards cells were fixed using an an ice-cold 70% [v/v] EtOH solution and incubated for 1 h on on ice.Following an an additional washing step with PBS, cells were incubated with FACS-stain solution (0.5% Triton X-100, 20 20 µg/ml PI, 20 20 µg/ml RNase A in in PBS) for 1 h to to stain the DNA and digest remaining RNA.DNA content was measured by by flow cytometry.

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Cells were plotted with the FSC-A against the SSC-A channel.The living population was gated and then plotted for the PI-A signal as as a histogram.Only PI PI positive cells were gated.G1, S and G2 G2 phases were determined based on on the PI PI signal.The same gates were transferred to to all samples in in the same experiment.
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